Investigating the impact of long term exposure to chemical agents on the chromosomal radiosensitivity using human lymphoblastoid GM1899A cells

This study aimed to investigate the impact of chronic low-level exposure to chemical carcinogens with different modes of action on the cellular response to ionising radiation. Human lymphoblastoid GM1899A cells were cultured in the presence of 4-nitroquinoline N-oxide (4NQO), N-nitroso-N-methylurea (MNU) and hydrogen peroxide (H2O2) for up to 6 months at the highest non-(geno)toxic concentration identified in pilot experiments. Acute challenge doses of 1 Gy X-rays were given and chromosome damage (dicentrics, acentric fragments, micronuclei, chromatid gaps/breaks) was scored. Chronic exposure to 20 ng/ml 4NQO, 0.25 μg/ml MNU or 10 μM H2O2 hardly induced dicentrics and did not significantly alter the yield of X-ray-induced dicentrics. Significant levels of acentric fragments were induced by all chemicals, which did not change during long-term exposure. Fragment data in combined treatment samples compared to single treatments were consistent with an additive effect of chemical and radiation exposure. Low level exposure to 4NQO induced micronuclei, the yields of which did not change throughout the 6 month exposure period. As for fragments, micronuclei yields for combined treatments were consistent with an additive effect of chemical and radiation. These results suggest that cellular radiation responses are not affected by long-term low-level chemical exposure.


Results and discussion
Initial statistical analyses. There was no evidence of departure from normality for any of the endpoints.
Pilot studies. Acute genotoxic effects of chemicals. For the initial identification of suitable chemical concentrations for long-term exposure of lymphoblastoid GM1899A cells, the micronucleus assay was used to determine the highest concentration that does not induce a significant increase of micronuclei after exposure to chemical for 24 h. The yield of micronuclei provided a good estimate of the overall genotoxic effect while the percentage of binucleated cells reflected how many cells passed through mitosis into the cytochalasin B-induced cytokinesis block during the chosen time and thus provided an indirect measure of cell proliferation. For 4NQO, Fig. 1a shows a steep reduction in the percentage of binucleated cells (p < 0.001).
Although the effect of chemical was found to be highly significant overall in terms of micronuclei induction (p < 0.001) a concentration of 20 ng/ml did not induce significant levels of micronuclei in binucleated cells (p = 0.060). The concentration of 20 ng/ml (0.06 μM) 4NQO was chosen for long-term exposures.
Hydrogen peroxide induced significant levels of micronuclei (p = 0.001) above a concentration of 5 μM (Tukey's test for comparison with 0 μM, p = 0.023), with a small but significant effect on cell cycle progression (p = 0.005; Fig. 1b).
Medium term (2 weeks) exposures to chemicals. GM1899A cells were initially cultured for two weeks in the presence of 20 ng/ml 4NQO, 10 μM H 2 O 2 or 20 μg/ml MNU , to confirm whether these concentrations were suitable for long-term exposure experiments. The treatment with 4NQO or H 2 O 2 was well tolerated over the two week pilot experiment period, causing only a slight (non-significant, ANOVA p for these treatments compared to control = 0.163) reduction in cell production rate (Fig. 2a). During the initial two-week exposure experiment, samples were taken every 3.5 days for flow cytometric analysis of cell cycle distribution changes. Minor, non-significant fluctuations of the cell cycle distribution were recorded both in controls and 4NQO-or H 2 O 2 -exposed cultures ( Fig. 2b; p > 0.999). Exposure to 20 μg/ml MNU, however, dramatically reduced the fraction of cells in G1 observed following 3.5 and 7 days of exposure (Fig. 2b). This indicates that cell cycle progression may be   53 . This delayed effect was observed in cell viability measurements (Fig. 3). Subsequently, to determine concentrations of MNU that would be compatible with long-term exposure scenarios, cell viability tests were performed for a range of concentrations. Figure 3 shows that even at a concentration of 2 μg/ml, less than 50% of cells were viable three days after the exposure, despite good viability at 24 h. Doses ranging from 0.1 to 1 μg/ml demonstrated a decrease in viability from 95 to 73% with increasing dose at 48 h, but a significantly steeper decrease from 91 to 51% at 72 h. Over the dose ranges assessed the dose of 0.25 μg/ml MNU was well tolerated by the cells at all three time points measured and was therefore subsequently used for long-term exposures.
Main study: long-term exposures to chemicals. GM1899A cells were chronically exposed to 20 ng/ml 4NQO, 10 μM H 2 O 2 , 0.25 μg/ml MNU or sham-exposed in a set of long-term experiments. Cells were split and medium and chemical renewed every 3.5 days. Samples of cells from each chemical exposure were exposed to 1 Gy of X-rays every four weeks and processed for 1) flow cytometry to monitor changes in cell cycle distribution and apoptosis, 2) chromosome aberration analysis and 3) the micronucleus assay.   Fig. 4a show that exposure to 20 ng/ml 4NQO was well tolerated over the 6 months period, with no significant effect of the chemical on cell numbers over time (p = 0.883), although the treatment resulted in a consistent 20% reduction in cell numbers for treated cells over the time period (p < 0.001). Exposure to 10 μM H 2 O 2 ( Fig. 4b) or to 0.25 μg/ ml MNU (Fig. 4c) was similarly well tolerated over the 6 months period, though both the chemical and time did induce a small, but significant, reduction in cell numbers during this time (p all < 0.001).
Long-term exposure to 20 ng/ml 4NQO induced a few dicentric chromosomes ( Fig. 5a; p = 0.004) and significant levels of acentric chromosome fragments ( Fig. 6a; p < 0.001) above the levels found in sham-exposed cells after months 1-6 of exposure, with a significant difference in the responses for the different time points for fragments only (p = 0.038). Direct induction of dicentrics by 4NQO would be somewhat unexpected, as this is mechanistically difficult to explain and has not been reported previously. However, the dicentrics observed here are likely 'derived' ones. Such indirectly induced dicentrics are typically not accompanied by an acentric fragment. They are formed when unrepaired DNA lesions are converted into chromosomal aberrations during DNA replication.
Exposure to 1 Gy X-rays induced similar yields of dicentrics at all time points, irrespective of whether cells had been chronically exposed to 4NQO or not, with significant induction associated with exposure to X-rays only (p < 0.001) but with no significant interaction effect detected for the X-rays plus the chemical (p = 0.175). Pooling of all time points was therefore justified since no time dependent changes were observed (see Supplementary  Table S1 online; p for time, all endpoints > 0.05). In total, 5 dicentrics and 11 acentrics were observed in 257 untreated cells, compared to 13 dicentrics and 57 acentrics in 300 4NQO-treated cells, whilst 310 cells exposed to 1 Gy X-rays contained 74 dicentrics/51 acentrics and 361 combined-treated cells contained 108 dicentrics/91 acentrics. See Supplementary Table S1 for the full data set on chromosomal aberrations.
The yields of chromosome damage did not change significantly for any of the treatment groups during long-term exposure. Exposure to 20 ng/ml 4NQO increased micronuclei levels consistently above the baseline levels and in combination with X-rays induced a significant increase in micronuclei compared with X-rays alone ( Fig. 7a and Supplementary Table S2; p < 0.001). This was consistent with an additive effect. As observed for chromosome aberrations, micronuclei formation did not change over the duration of exposure (p = 0.560) so that individual counts were pooled to give 54 micronuclei in 1407 binucleated untreated cells; 149 in 1310 X-irradiated cells; 122 in 1248 4NQO-treated cells and 189 in 1195 combined-treated cells.
Long-term exposure to 10 μM H 2 O 2 did not induce any dicentric chromosomes (Fig. 5b) but a similar number of chromosome fragments as 1 Gy of X-rays (Fig. 6b) above the level found in sham-exposed cells after long-term exposure (p < 0.05). Exposure to 1 Gy X-rays induced a similar level of dicentrics at all time points, irrespective of whether cells had been chronically exposed to hydrogen peroxide or not. Chromosome fragment data show a slight, but not significant, increase in combined treatment samples compared to single treatments, consistent with an additive effect.
Exposure to 10 μM H 2 O 2 increased micronuclei levels slightly, but consistently above the baseline levels and in combination with X-rays induced a significant increase in micronuclei compared with X-rays alone ( Fig. 7b and Supplementary Table S2; p < 0.001). This was consistent with an additive effect. Micronuclei formation did not change over the duration of exposure so that individual counts were pooled to give 50 micronuclei in 1265  Figure 5c shows that exposure to 0.25 μg/ml MNU did not induce a significant number of dicentrics but levels of chromosome fragments shown in Fig. 6c were similar to the samples treated with 1 Gy of X-rays and sham-exposed cells after 5 months of exposure. Exposure to 1 Gy X-rays induced a similar level of dicentrics at all time points, but levels tended to be slightly higher for cells that had been chronically exposed to MNU. This trend was, however, not significant. Chromosome fragment data show an increase in combined treatment samples compared to single treatments, consistent with an additive effect. Figure 7c shows that exposure to 0.25 μg/ml MNU increased somewhat the micronuclei levels and in combination with X-rays induced a significant increase in micronuclei compared with X-rays alone (for all data set see Supplementary Table S2; p < 0.001). Individual counts for each treatment group were pooled because micronuclei formation did not change over the 5 months duration of exposure. 49 micronuclei were observed in 1220 binucleated untreated cells; 141 in 1250 X-irradiated cells; 118 in 1250 MNU-treated cells and 167 in 1120 combined-treated cells.
Our in vitro studies using human EBV-transformed lymphoblastoid cells have aimed to simulate exposure situations in which an individual is exposed chronically to a chemical genotoxicant and is then exposed to ionising radiation in a medical or accidental context.
In conclusion no significant acute effects on cell proliferation from any of the three agents was observed at the concentrations selected for chronic exposures, however, there was induction of various types of chromosomal damage such as micronuclei, dicentrics, and fragments-manifestations of chromosome injury which also are www.nature.com/scientificreports/ typical for radiation exposure. The crucial question is whether chronic exposure to these chemicals sensitises cells to subsequent radiation exposure or whether effects from either are just additive. A similar study has been performed on sodium arsenite, which is a well-established carcinogen and genotoxicant using the same experimental protocol and the same model 25 . Here other genotoxic agents have been investigated, results of these studies are as summarised above.
The consistent finding of additive effects for the studied combined exposures suggests that, typically, radiation responses assessed with cytogenetic end points do not seem to be altered by long-term low level chemical exposure of GM1899A cells. Specifically, no significant adaptive responses or sensitising effects were observed in this cell line. Instead, the cellular response mechanisms for radiation damage, DNA repair, cell cycle checkpoint control and cellular survival/death pathways, seem to operate without any modulatory effects from chronic lowlevel chemical exposure in the systems analysed here. The obvious limitation of this study is the in vitro nature of the work which involved the use of an established human EBV-transformed lymphoblastoid cell line rather than primary human tissues. Therefore, this study is in itself insufficient to provide conclusive evidence on human health implications from combined exposures. The above conclusions are only valid for ionising radiation as a challenging agent and may depend on the use of this particular cell line. In vivo, supracellular and systemic aspects like inflammatory and immune responses need to be taken into account in future work. Although, the cytogenetic endpoints used here are currently the most suitable biomarkers for cancer risk, they are probably not informative for non-cancer effects like cardiovascular diseases which have more recently emerged as important medical conditions associated with exposure to low or moderate levels of a range of environmental hazards, including chemicals and ionising radiation 54 . Integration of sublethal concentrations and endpoints into risk assessment frameworks is important. This study does not deal with chronic toxicity effects arising at individual or population levels following long-term continuous or fluctuating exposure to chemicals at sublethal concentrations (not high enough to cause mortality or directly observable impairment following acute short-term exposure).
There are many examples of studies on combined exposures to ionising radiation and genotoxic agents including the improvement of tumour therapy by concurrent treatment with a chemical. However, the high doses and deterministic effects involved in these combined therapies cannot be simply related to low level combined effects.
We conclude, in agreement with the UNSCEAR 2000 Report 2 that exposures of GM1899A cells to radiation and low level chemicals yield additive effects.

Materials and methods
Most of the procedures described here were the same as in Nuta et al. 25 .
Cells. GM 1899A, a normal human lymphoblastoid line (received from Dr M O'Donovan, Astra Charnwood, Loughborough, UK) was used for all experiments. Many studies on chemical exposures are performed using lymphoblastoid cells lines such as TK6, AHH-1 8,39,40 . Usage of lymphoblastoid cell lines established by in vitro infection with Epstein Barr Virus (EBV) as a reliable model system for carcinogen sensitivity, DNA damage/ repair and other analyses has been consistent throughout the last decade. Since this is a study in which acute and long term (6 months) toxicity of the genotoxins were tested there was a reason to believe that the GM1899A cell line that was readily available in our lab is in close resemblance with the parent lymphocytes. Cells were grown in suspension at 37 °C in a humidified atmosphere of 95% air: 5% CO 2 in Dutch Modified RPMI 1640 medium supplemented with 20% heat-inactivated fetal bovine serum, 2 mM sodium pyruvate, 2 mM l-glutamine and antibiotic/antimycotic (penicilin streptomycin solution)(Gibco Life Technologies, UK). For all experiments, asynchronous suspension cultures in the exponential phase of growth were seeded from the same batch and grown in T75 flasks.

Treatment with chemicals. N-nitroso-N-methylurea (MNU) and 4-nitroquinoline-1-oxide (4NQO)
were initially dissolved in DMSO and then further diluted in water. Hydrogen peroxide (H 2 O 2 ) was diluted in water. Chemicals were supplied by Sigma-Aldrich, UK. DMSO concentrations were never higher than 0.1% for acute exposures and below 0.01% for chronic exposures. For acute exposures, cells seeded at 0.6 × 10 5 cells/ml were treated with different concentrations of chemicals for 24 h. For long-term exposures, cells were cultured in the presence of 0 or 20 ng/ml 4NQO, 0 or 0.25 μg/ml MNU, and 0 or 10 μM H 2 O 2 . Cells were counted, split and medium and chemical renewed every 3.5 days.
Irradiation. Every four weeks, aliquots of long-term chemical-treated cells were exposed at room temperature to 0 or 1 Gy of 250 kVp X-rays, with 11 mA and Al/Cu filtration, at a dose rate of approximately 1 Gy/min. The non-chemical exposed parallel controls were also X-rayed and sham X-rayed. Physical dosimetry was carried out with a calibrated Farmer dosemeter in the same geometry as the specimens.
Cytokinesis-blocked micronucleus assay. Treated and sham-treated cells were cultured for 24 h in the presence of 3 μg/ml cytochalasin B (added 30 min post irradiation) and washed with medium. For the acute and the long-term exposures, cells were resuspended in 5 ml cold hypotonic solution (KCl 5.6 g/l), spun down again and fixed in 5 ml fixative (acetic-acid: methanol, 1:10 v/v) (Fisher Scientific, UK). Three drops of formaldehyde 37% (Polysciences, UK) were added and the fixative was changed twice after centrifugation at 600 rpm for 8 min. Fixed cell suspensions were dropped onto pre-cleaned microscope slides with a drawn-out Pasteur pipette and all slides were stained for 3.5-5 min in a 2% aqueous Giemsa solution (BDH Laboratory Supplies, UK), air-dried at room temperature and mounted in DPX mounting medium (Thermo Scientific, UK). Micronuclei in binucleated cells were scored by eye at 1000 × magnification with an Axioskop (ZEISS, Germany) microscope under oil immersion. www.nature.com/scientificreports/ Chromosomal aberration analyses. Colcemid at a concentration of 25 μg/ml was added to each cell culture 22 h after irradiation or mock-irradiation which was then returned to the incubator for 2 h. After this time cells were spun down, treated with prewarmed hypotonic KCl solution (5.6 g/l) and incubated for 15 min in a water bath at 37 °C, spun down and fixed three times in methanol: acetic acid (3:1 v/v). The slides were prepared and stained with Giemsa solution as described above. Scoring of dicentrics, acentric fragments and all other aberrations was carried out at 1,000 × magnification under oil immersion using the Metafer metaphase finding system (MetaSystems). All dicentrics were scored, noting whether or not they were accompanied by an acentric fragment. In addition, excess acentric fragments were scored, i.e. those not accompanied by a dicentric.
Flow cytometric cell cycle analyses. Cells were harvested by centrifugation at 1200 rpm at 4 °C for 5 min and resuspended in PBS. 10 6 cells were prepared per tube, fixed in cold ethanol (70%) and stained with 1 μg/ml propidium iodide solution. Cells were analysed in a FACSCalibur flow cytometer (BD Biosciences) with excitation at 488 nm.
MTT assay. The CellTiter-Blue cell viability assay (Promega) was used. Triplicate wells containing cells treated with MNU at different concentrations were set in parallel with no-cell control wells which served as negative control to determine background fluorescence and with untreated cells control wells. Briefly, 96-well plates containing cells in culture medium were prepared and the test compound and vehicle controls were set up according to the protocol. Cells were cultured for the desired test exposure period, removed from the incubator and 20 µl/well of CellTiter-Blue reagent was added to each plate. After an incubation step, fluorescence at 560/590 nm was recorded using a plate-reading fluorometer. Calculation of results was executed according to the suppliers' protocol.
Data analyses. The "Dose Estimate" software was used to calculate Poisson errors 55 .
A subset of the control and treated data for each analysis and endpoint were tested for normality using the Anderson Darling normality testing. General linear model analysis of variance (ANOVA) was then carried out to investigate the effects of the experimental factors on the outcomes. For the pilot studies, for acute genotoxic effects of chemicals, the ANOVA model factor was chemical concentration and the response was percentage of binucleated cells or yield of micronuclei. For the medium term exposures to chemicals, the model factors were culture time and chemical for the response relative cell number, and concentration and time for the response percentage viability. For the main study, ANOVA was used to assess the impact of factors days in culture (0-160) with or without the chemicals on cell count. To investigate the role of radiation with or without chemicals on the observed number of dicentrics per metaphase, fragments per metaphase and total micronuclei per binucleated cell, the ANOVA factors were exposure month (1-6), radiation dose (0 or 1 Gy), and chemical (0 or concentrations as detailed in the main text). In each case, the effect of the irradiation and chemicals was also tested for evidence of interaction between these factors.

Data availability
All data generated and analysed during this study are included in this published article (and its Supplementary Information files).